title: Obatoclax induces Atg7-dependent autophagy independent of beclin-1 and BAX/BAK.
abstract: Direct pharmacological targeting of the anti-apoptotic B-cell lymphoma-2 (BCL-2) family is an attractive therapeutic strategy for treating cancer. Obatoclax is a pan-BCL-2 family inhibitor currently in clinical development. Here we show that, although obatoclax can induce mitochondrial apoptosis dependent on BCL-2 associated x protein/BCL-2 antagonist killer (BAX/BAK) consistent with its on-target pharmacodynamics, simultaneous silencing of both BAX and BAK did not abolish acute toxicity or loss of clonogenicity. This is despite complete inhibition of apoptosis. Obatoclax dramatically reduced viability without inducing loss of plasma membrane integrity. This was associated with rapid processing of light chain-3 (LC3) and reduction of S6 kinase phosphorylation, consistent with autophagy. Dramatic ultrastructural vacuolation, not typical of autophagy, was also induced. Silencing of beclin-1 failed to prevent LC3 processing, whereas knockout of autophagy-related (Atg)7 abolished LC3 processing but failed to prevent obatoclax-induced loss of clonogenicity or ultrastructural changes. siRNA silencing of Atg7 in BAX/BAK knockout mouse embryonic fibroblasts did not prevent obatoclax-induced loss of viability. Cells selected for obatoclax resistance evaded apoptosis independent of changes in BCL-2 family expression and displayed reduced LC3 processing. In summary, obatoclax exhibits BAX- and BAK-dependent and -independent mechanisms of toxicity and activation of autophagy. Mechanisms other than autophagy and apoptosis are blocked in obatoclax resistant cells and contribute significantly to obatoclax's anticancer efficacy.
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, Northern Ireland, UK.
authors.forename: F
authors.initials: F
authors.lastname: McCoy
authors.forename: J
authors.initials: J
authors.lastname: Hurwitz
authors.forename: N
authors.initials: N
authors.lastname: McTavish
authors.forename: I
authors.initials: I
authors.lastname: Paul
authors.forename: C
authors.initials: C
authors.lastname: Barnes
authors.forename: B
authors.initials: B
authors.lastname: O'Hagan
authors.forename: K
authors.initials: K
authors.lastname: Odrzywol
authors.forename: J
authors.initials: J
authors.lastname: Murray
authors.forename: D
authors.initials: D
authors.lastname: Longley
authors.forename: G
authors.initials: G
authors.lastname: McKerr
authors.forename: D A
authors.initials: DA
authors.lastname: Fennell
mesh_terms: D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000502:physiology; D001343:Autophagy / Q000187:drug effects*; D000071193:Autophagy-Related Protein 7; D000071186:Beclin-1; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D006801:Humans; D007211:Indoles; D008565:Membrane Proteins / Q000502:physiology; D051379:Mice; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011758:Pyrroles / Q000494:pharmacology*; D044764:Ubiquitin-Activating Enzymes / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D007211:Indoles; D008565:Membrane Proteins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2; D011758:Pyrroles; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; C509349:ATG7 protein, human; D000071193:Autophagy-Related Protein 7; D044764:Ubiquitin-Activating Enzymes; C520962:obatoclax